The silkworm (Bombyx mori) gut microbiota is involved in metabolic detoxification by glucosylation of plant toxins

Herbivores have evolved the ability to detoxify feed components through different mechanisms. The oligophagous silkworm feeds on Cudrania tricuspidata leaves (CTLs) instead of mulberry leaves for the purpose of producing special, high-quality silk. However, CTL-fed silkworms are found to have smaller bodies, slower growth and lower silk production than those fed mulberry leaves. Here, we show that the high content of prenylated isoflavones (PIFs) that occurred in CTLs is converted into glycosylated derivatives (GPIFs) in silkworm faeces through the silkworm gut microbiota, and this biotransformation is the key process in PIFs detoxification because GPIFs are found to be much less toxic, as revealed both in vitro and in vivo. Additionally, adding Bacillus subtilis as a probiotic to remodel the gut microbiota could beneficially promote silkworm growth and development. Consequently, this study provides meaningful guidance for silk production by improving the adaptability of CTL-fed silkworms.

The manuscript by Hongxiang Lou and co-authors investigates the capacity of the silkworm gut microbiome to biotransform prenylated isoflavones (PIFs) into less toxic glycosylated derivatives (GPIFs). CTLs are highly concentrated in Cudrania tricuspidata leaves, which is fed to the silkworm in order to strengthen the quality of produced silk. The authors analysed the content of isoflavones in the silkworm diet and faeces, and concluded that the gut microbiota, and in particular Bacillus sp. contribute to the isoflavones glycosylation, what provides a detoxifying effect to the host, and promotes its better growth and development, essentially leading to the improved silk production. Major part of the manuscript is devoted to the chemical analysis of the metabolites in the diet and insect faeces with techniques such as HPLC, LC-MS and NMR. This part of study is very well described, in terms of the methos used and generated results, and extensive supplementary data is also provided. I acknowledge the authors for this detailed analysis! The second part investigates the gut microbiome basically through the community profiling with the use of the 16S rRNA gene amplicon sequencing. In addition, some in vitro studies on the toxicity of PIFs on isolated Bacillus species and the capacity of these bacteria to biotransform PIFs, as well as and a bio-augmentation of gut microbiota with probiotic Bacillus sp. were also proposed. The study is quite interdisciplinary, however a strong focus is given to the chemical analysis, while my expertise in this domain is rather limited, therefore I will more focus on the microbiome-related aspects. I hope the other reviewers provide a constructive revision of this part of the study. My major concern with regards to the microbiome analysis, is that there is actually a lack of evidence that the Bacillus species are the main microbes involved in PIFs biotransformation. I don't question their ability to do it, and the authors have showed it via the in vitro assays, and also by evidencing the beneficial effect on the host of gut microbiota bio-augmentation with Bacillus sp. However, in general, Bacillus sp. doesn't seem to dominate the gut microbiota of the silkworm (see Fig. 6). While other phyla that are much more abundant in the gut were not considered here at all. Also, most if not all bacteria encode in their genomes multiple glycosyltransferases. Therefore, a sentence in lines 123-125 doesn't bring real evidence that these species are key to the biotransforming activity of the gut microbiome. In my opinion the authors a priori decided that Bacillus sp are important microorganisms in the silkworm microbiome, contributing to the detoxification effect, and designed the whole experimental part around this assumption. This is not a big issue, however I would clearly indicate it in the manuscript that the microbial analysis was targeted towards the analysis of the detoxifying activity of Bacillus sp., and not the gut microbiome. In addition, there is also no proof, that Bacillus species present in the silkworm gut are the same or at least closely related to the strains employed in the in vitro tests. I could not spot any 16S rRNA gene sequence comparison pointing to this. I think this study is of general interest and might be of hight industrial relevance for silkworm breeders. Even though the scientific evidence on the establishment of the administrated probiotic Bacillus strain in the gut microbiome is relative week, the observed health beneficial effect of the probiotic powder used can have important applied implications. To my opinion, any statistical analysis used is correctly employed, however some concern about the microbiome characterisation is given below. More specific comments are listed below: 1. Overall, the manuscript is quite well written in English, however some sentences are a bit strangely phrased. I am not a native English speaker, so I can only give some advice here and suggest revising the whole manuscript accordingly. For example: lines 42-43, I guess the gut microbiota contributes to the protection against toxic compounds and not insects? Lines: 73-75, …one bacterium in a gut?? Lines 100-105: The sentence is spread over six lines, try breaking it into shorter sentences. Lines 118-120: Rewrite please, difficult to understand the conclusion. Line 122: Microbial biotransformation by Bacillus and not of Bacillus?. etc… 2. Lines 123-127: Rewrite to state your hypothesis in a way it directly targets Bacillus as a group of interest in this study. It should be written that there might be other microbes contributing to the process of biotransformation, however they were not investigated here. You also cite Fig.6  3. Line 128: Again, according to Fig. 6, there is little evidence that Bacillus would be the dominant microbe in the silkworm gut. 4. Lines 129-130: Is this your conclusion, or a citation of another study? If the latter, please include the reference. 5. Lines 137-138: This cannot be concluded, as other gut microbes were not examined in this study. Also, no real Bacillus isolate from the silkworm gut was characterised, neither in vivo, nor in vitro (e.g. using metagenomics or metatranscriptomics). Therefore, we can only speculate here that based on the analysis of previously characterised Bacillus sp., silkworm gut Bacillus could also contribute to the PIFs biotransformation. 6. Lines 144-146: I would rather say that you evidenced indirectly the contribution of Bacillus sp to the toxic compound biotransformation, but we cannot exclude that other microbes can be even more active in this process. 7. Line 160: I would rather say that "..1-5 was rather weak or..", than "greatly weak". 8. Lines 162-164: Not sure we can say that the growth was in good condition, consider rephrasing the sentence. 9. Line 175: 50% survival "rate" and not 50% survival; consider revising accordingly. 10. Paragraph "B. subtilis as a probiotic influenced gut flora by 16S rDNA amplicon sequence analysis", lines 186-206. Have you checked if the bacterium established in the gut? I would be surprised to know that an isolate from a completely different environment than the silkworm gut, well integrates with the insect gut microbiota. Usually, these insect gut microbes have been evolving together with the host over the evolution, are host-specific and often found nowhere else than in this specific gut environment. You could perhaps compare the 16S rRNA sequences of the gut microbiota and the probiotic strain used, over the time (or at least before, and sometime after the probiotic administration, and also in comparison with the control group) 11. The same paragraph as above; consider revising the header. It reads as if the gut flora was influenced by the 16s rRDA analysis. 12. Not enough details are provided on the 16S rRNA gene amplicon sequencing and data analysis. For example, to say that "DNA samples were amplified" (line:382) is little specific. I would rather give the details on the protocol used, including the information on the primers used to amplify a specific 16S rRNA gene region. 13. Lines 211-217: I think that these two consecutive sentences give the same information, don't they? Please revise to avoid repetition. 14. The same paragraphs (lines 208-239). Is it possible that any component contained in the Bacillus probiotic powder could contribute to this effect? If not, please revise your 16S rRNA data to show any evidence that supplemented Bacillus sp integrated with the gut flora, providing this long-lasting beneficial effect to its host.
The manuscript was carefully revised according to the comments from reviewers. All changes in this manuscript and supplemental material have been highlighted. The changes are detailed in the document of "Point by point response to reviewers".

For Reviewers' Comments:
For Reviewer 1: Q1: You should analyse whether the isoflavonoids are taken up by the isnect larvae; check if you find them in the integument or body tissues. It could be that a detoxification also occurs inside the insect and not only my gut microbes.
Response: Thanks for your kind suggestions. We focused the detoxification metabolism by silkworm intestinal microbiota. About the components in the insect tissue, we also tested the distribution by HPLC-MS in supplementary Fig. S218, we found DPL (24) and the corresponding GPIFs were all present in silkworm integument, silk gland, midgut and body fluid, we cannot deny the possibility that the detoxification partially occurred inside the silkworm. Q4. The presentation and discussion of the microbiome data are inadequate.
Response: Thanks for your kind suggestions. We have further discussed the microbiome data and revised this section in the revised version.
Q5. The English needs improvement by a native speaker.
Response: Thanks for your kind suggestion. The language of this paper has been sent for scientific editing by Springer Nature Author Services. The language editing certificate is attached as an SI file.
For Reviewer 2: Q1: Overall, the manuscript is quite well written in English, however some sentences are a bit strangely phrased. I am not a native English speaker, so I can only give some advice here and suggest revising the whole manuscript accordingly. For example: lines 42-43, I guess the gut microbiota contributes to the protection against toxic compounds and not insects? Lines: 73-75, …one bacterium in a gut?? Lines 100-105: The sentence is spread over six lines, try breaking it into shorter sentences. Lines 118-120: Rewrite please, difficult to understand the conclusion. Line 122: Microbial biotransformation by Bacillus and not of Bacillus. etc.
Response: Thanks to the comment from you. We have checked the text carefully according to your suggestions and corrected it accordingly.
Q2: Lines 123-127: Rewrite to state your hypothesis in a way it directly targets Bacillus as a group of interest in this study. It should be written that there might be other microbes contributing to the process of biotransformation, however they were not investigated here. You also cite Fig.6 before Fig. 3-5 are actually cited. I think this should be corrected. The same refers to the citation of Fig.3 (line 97), I think it goes before Fig. 2 (line 103). Consider revising the whole manuscript accordingly. Q4: Lines 129-130: Is this your conclusion, or a citation of another study? If the latter, please include the reference.
Response: Thanks for your kind suggestions. In the isolation experiment of silkworm gut microbiota, we found that the B. subtilis isolate was as the dominant flora in silkworm gut. Q5-Q6: Lines 137-138: This cannot be concluded, as other gut microbes were not examined in this study. Also, no real Bacillus isolate from the silkworm gut was characterised, neither in vivo, nor in vitro (e.g. using metagenomics or metatranscriptomics). Therefore, we can only speculate here that based on the analysis of previously characterised Bacillus sp., silkworm gut Bacillus could also contribute to the PIFs biotransformation. Lines 144-146: I would rather say that you evidenced indirectly the contribution of Bacillus sp to the toxic compound biotransformation, but we cannot exclude that other microbes can be even more active in this process.
Response: Thanks for your kind suggestions. We took time, isolated and obtained a Bacillus subtilis strain, and demonstrated that B. subtilis existed in silkworm gut and added the related data in the present version. We also have demonstrated the conversion capacity of B. subtilis strain isolated in vitro ( Fig. 4b (iii)). Q7: Line 160: I would rather say that "..1-5 was rather weak or..", than "greatly weak".
Response: Thanks for your kind suggestions. We have corrected it accordingly.
Q8: Lines 162-164: Not sure we can say that the growth was in good condition, consider rephrasing the sentence.
Response: Thanks for your kind suggestions. We have rephrased it accordingly. Q9: Line 175: 50% survival "rate" and not 50% survival; consider revising accordingly.
Response: Thanks for your kind suggestions. We have corrected it accordingly. Q10: Paragraph "B. subtilis as a probiotic influenced gut flora by 16S rDNA amplicon sequence analysis", lines 186-206. Have you checked if the bacterium established in the gut? I would be surprised to know that an isolate from a completely different environment than the silkworm gut, well integrates with the insect gut microbiota. Usually, these insect gut microbes have been evolving together with the host over the evolution, are host-specific and often found nowhere else than in this specific gut environment. You could perhaps compare the 16S rRNA sequences of the gut microbiota and the probiotic strain used, over the time (or at least before, and sometime after the probiotic administration, and also in comparison with the control group) Response: Thanks for your kind suggestions. Here, we have isolated and obtained Bacillus subtilis strain and demonstrated that Bacillus subtilis existed in silkworm gut in the revised version. Q11: The same paragraph as above; consider revising the header. It reads as if the gut flora was influenced by the 16s rRDA analysis.
Response: Thanks for your kind suggestions. We have revised it accordingly. Q12: Not enough details are provided on the 16S rRNA gene amplicon sequencing and data analysis. For example, to say that "DNA samples were amplified" (line:382) is little specific. I would rather give the details on the protocol used, including the information on the primers used to amplify a specific 16S rRNA gene region.
Response: Thanks for your kind suggestions. We have supplemented this section and the details provided on the 16S rDNA gene amplicon sequencing and data analysis in supplementary S7. Q13: Lines 211-217: I think that these two consecutive sentences give the same information, don't they? Please revise to avoid repetition. Q14: The same paragraphs (lines 208-239). Is it possible that any component contained in the Bacillus probiotic powder could contribute to this effect? If not, please revise your 16S rRNA data to show any evidence that supplemented Bacillus sp integrated with the gut flora, providing this long-lasting beneficial effect to its host.
Thank you very much for your attention and consideration for our manuscript entitled "The silkworm gut microbiota involved in metabolic detoxification by glucosylation of plant toxins". In order to better answer the questions, we added the relevant experiments of the isolation and identification of silkworm intestinal microbes to the revised article.
According to the request of the email, we have carefully addressed the remaining concerns from Reviewer #2 as follows. All changes in this manuscript and supplemental material have been highlighted.

For Reviewers' Comments:
For Reviewer 2: Q3: Line 128: Again, according to Fig. 6, there is little evidence that Bacillus would be the dominant microbe in the silkworm gut.
Response: Thanks for your kind suggestions. Although it was reported in previous studies that Bacillus is one of the dominant microbes in silkworm gut, we did not make a conclusion of Bacillus as dominant microbe in the silkworm gut by Fig.6. We actually proved the presence of Bacillus in silkworm gut by Fig. 6, and further demonstrate the changes of the intestinal microbes of the experimental group compared with the control group after adding Bacillus subtilis as a probiotic. In addition, we have demonstrated Bacillus in silkworm gut contributed to the PIFs biotransformation in vitro, which suggested that Bacillus also played important role in the silkworm gut. To get our point across more accurately, we have revised the description of this section in the manuscript in Line 133, 154-160, 308. Q5-Q6: Lines 137-138: This cannot be concluded, as other gut microbes were not examined in this study. Also, no real Bacillus isolate from the silkworm gut was characterised, neither in vivo, nor in vitro (e.g. using metagenomics or metatranscriptomics). Therefore, we can only speculate here that based on the analysis of previously characterised Bacillus sp., silkworm gut Bacillus could also contribute to the PIFs biotransformation. Lines 144-146: I would rather say that you evidenced indirectly the contribution of Bacillus sp to the toxic compound biotransformation, but we cannot exclude that other microbes can be even more active in this process.
Response: Thanks for your kind suggestions. Firstly, to better reveal the effects of gut bacteria, we have isolated the intestinal bacteria of silkworm by NA medium plate (in Line 411-418) and three isolates were obtained and identified as Bacillus subtilis, Staphylococcus sciuri and Enterobacter hormaechei strain, respectively. Then, we further studied the microbial transformation in vitro by three strains from silkworm gut of DPL (24). And we also found that the glycosylation ability of B.
subtilis from silkworm gut for DPL (24) was more potent than that of the other two strains ( Supplementary Fig. S219). B. subtilis could glycosylate DPL (24) to form 1-5, however, S. sciuri and E. hormaechei could only partially convert DPL (24) to produce corresponding GPIFs (Supplementary S219). Therefore, it suggested that the formation of abundant GPIFs in SWFs was closely related to silkworm intestinal bacteria. And we can evidence the direct contribution of Bacillus sp. with potent glycosylation capacity of DPL (24) in silkworm gut to the toxic compound biotransformation, but we cannot determine whether Bacillus sp. would be the most active in this process. All above mentioned was seen in Line 122-130, 154-160. Q10: Paragraph "B. subtilis as a probiotic influenced gut flora by 16S rDNA amplicon sequence analysis", lines 186-206. Have you checked if the bacterium established in the gut? I would be surprised to know that an isolate from a completely different environment than the silkworm gut, well integrates with the insect gut microbiota. Usually, these insect gut microbes have been evolving together with the host over the evolution, are host-specific and often found nowhere else than in this specific gut environment. You could perhaps compare the 16S rRNA sequences of the gut microbiota and the probiotic strain used, over the time (or at least before, and sometime after the probiotic administration, and also in comparison with the control group) Q12: Not enough details are provided on the 16S rRNA gene amplicon sequencing and data analysis. For example, to say that "DNA samples were amplified" (line:382) is little specific. I would rather give the details on the protocol used, including the information on the primers used to amplify a specific 16S rRNA gene region.
Response: Thanks for your kind suggestions. We have supplemented this section and the details provided on the 16S rDNA gene amplicon sequencing and data analysis in supplementary S7 in Line 450-487. Q14: The same paragraphs (lines 208-239). Is it possible that any component contained in the Bacillus probiotic powder could contribute to this effect? If not, please revise your 16S rRNA data to show any evidence that supplemented Bacillus sp integrated with the gut flora, providing this long-lasting beneficial effect to its host.
Response: Thanks for your kind suggestions. The Bacillus subtilis powder used in our work was a single strain, and no other bacteria were incorporated into the preparation process. Besides, to better demonstrate that any component contained in the Bacillus probiotic powder did not affect the glycosylation, the Bacillus powder sterilized at 120℃ for 30min was used to biotransformation of DPL (24) in vitro, and the formation of corresponding glycosylation products could not be detected by LC-MS. Any component contained in the Bacillus probiotic powder had no effect on the experiment of biotransformation in vitro, but whether they have an effect in vivo has not been studied. In addition, according to our 16S rDNA data, it increased the relative abundance of the genus Bacillus in the silkworm gut after adding B. subtilis probiotic, which increased from 1.19% to 2.12% in YSY-silkworm, increased from 0.72% to 1.26% in HK2-silkworm (Fig. 6c). Furthermore, it also increased the gut microbial diversity and overall evenness. And we have revised and discussed the microbiome data in the last revised version and this revised version in Line 209-220.